Molecular Mechanism Of Brucella UGPase Regulation Of Host Molecule ACOD1 Expression And Consequently Inhibition Of NF-κB Activation

Brucellosis("brucellosis")is a serious zoonotic infection caused by Brucella spp.and is ranked as the top of Class B infectious diseases among the statutory infectious diseases in China.The disease is prevalent worldwide and has seriously affected the healthy development of animal husbandry and public health security.However,the prevention,control and decontamination of Brucella are seriously hampered by the fact that the function of some important virulence molecules of Brucella and the mechanism of escape from the immune response after infection of host cells are not fully understood.To this end,we investigated the proteins regulated by the new virulence factor UGPase of Brucella abortus that are differentially expressed in RAW264.7 cells,with a view to explaining the virulence function of UGPase from a host molecular perspective.First,the Label-free results were validated and analyzed using PRM protein quantification and WB techniques,based on comparative proteomic analysis of RAW264.7 cells infected before and after Brucella UGPase deletion using Label-free technology in the pre-laboratory.PRM protein quantification technology showed that UGPase up-regulated the expression of 8 protein molecules and down-regulated the expression of 13 protein molecules,which was completely consistent with the Label-free results.Based on the results of PRM protein quantification,the differential host molecules Rhoa,Cox2,S100a4and ACOD1 were selected again for validation by WB technique,and the results showed that UGPase promoted S100a4 expression,while suppressed Rhoa,Cox2 and ACOD1 expression,in full agreement with PRM protein quantification and Label-free results.It shows that the comparative proteomics and PRM protein quantitative results are reliable.Secondly,we selected the above-validated differentially expressed host molecule ACOD1 and constructed overexpressing ACOD1 cell lines using lentiviral packaging technology.Total RNA from RAW264.7 cells was reverse transcribed as c DNA as template,and the ACOD1 fragment was obtained by PCR amplification technique and then ligated to the empty lentiviral expression vector p LV-CMV-EGFP/Puro using NEBuilder seamless ligase.The above recombinant vector was transduced to 293T by calcium phosphate precipitation method for lentiviral packaging preparation,and a virus titer of 1.00×10~8TU/m L was obtained.RAW264.7 cells in good growth condition were selected for infection experiments with MOI of 10.After 48 h,continuous screening cultures were performed for about 1week at the optimal screening concentration of 3μg/m L of puromycin for RAW264.7 cells determined by the pre-experiment.After limited dilution of the surviving cells from the screening and expanded culture,the resulting single cell line was named RAW264.7-EGFP/Puro-ACOD1.The RAW264.7-EGFP/Puro-ACOD1 cell line was characterized using fluorescence microscopy,flow cytometry and WB.A stable fluorescence signal was detected by fluorescence microscopy,and no significant changes in cell morphology and activity were observed.Flow cytometry showed RAW264.7-EGFP/Puro-ACOD1 fluorescence rate of 97.17%.WB technique showed that ACOD1protein expression was extremely significantly higher in RAW264.7-EGFP/Puro-ACOD1 cell line than in RAW264.7 cells.Taken together,the above statements indicate the successful establishment of the RAW264.7 cell line overexpressing ACOD1 protein.Finally,the above construct of RAW264.7 cell line overexpressing ACOD1 protein was used as a tool to perform stimulation experiments using LPS and divided into 2 time points of 12 h and 24 h.NF-κB activation and cytokine expression were detected by WB and q PCR techniques,respectively.WB results showed that ACOD1 was able to promote the activation of NF-κB signaling pathway in host cells,but the promotion intensity was significantly weakened with increasing time.The q PCR results showed that ACOD1 up-regulated IFN-γexpression and down-regulated factors such as IL-1β,IL-2 and IL-10.In summary,we demonstrated that UGPase can help Brucella evade host immune surveillance for intracellular survival by downregulating host molecule ACOD1 expression,which in turn inhibits host NF-κB pathway activation and thus regulates cytokine expression to exert a virulence effect.